EVALUATION OF COMPETITIVE ADHERENCE OF LACTOBACILLUS

SPECIES ISOLATED FROM NUNU AGAINST SHIGELLA

BY

EZECHIYERE CHIAMAKA BERNICE

REG NO: 18286118

SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF BACHELOR OF SCIENCE (B.Sc.) DEGREE TO
THE DEPARTMENT OF MICROBIOLOGY, FACULTY OF SCIENCES,
UNIVERSITY OF ABUJA.

AUGUST, 2023.

1
 DEDICATION

I dedicate this project to God Almighty.

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 DECLARATION

I hereby declare that this work is the product of my own research efforts;

undertaken under the supervision of Prof. B.U. Akanbi and has not been presented

and will not be presented elsewhere for the award of a degree or certificate. All

sources has been duly acknowledged.

__________________________________

EZECHIYERE CHIAMAKA BERNICE

18286118

CERTIFICATION

3
This project report titled Evaluation of Competitive adherence of lactobacillus
species isolated from nunu against Shigella by Ezechiyere Chiamaka Bernice. Reg
No: 18286118 meets the regulations governing the award of the degree of Bachelor
of Science of the University of Abuja.

_____________________ ____________________

Dr. B. O Akanbi Date

Supervisor

_____________________ ___________________
Dr. S. S. Machunga-Mambula Date
Head of Department

_____________________ ___________________
Dr. Ahmad Belgore Date
Plagiarism Checker

_____________________ ___________________
External Examiner Date

ACKNOWLEDGEMENTS

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I remain grateful to Almighty GOD for his infinite mercies, love and kindness

toward me and for making my project work comes to reality.

I am highly indebted to my supervisor, Dr. B.O Akanbi, who supervised this

work despite his busy schedule, I must commend his simplicity and discipline

when it comes to work. I have truly learned from him not as a lecturer to student

but as a daughter would learn from a father, may God Almighty bless him in an

immeasurable way.

Special thanks goes to the Head of Department Prof. S.S. Machunga- Mambula and

all other lecturers who have impacted my life positively during the cause of my

study in this great institution.

My special gratitude goes to my strong and courageous parent, Mr. and Mrs.

Ezechiyere for their prayers, financial support and encouragement through this

program. May you eat the fruit of your labor.

Finally, I also want to appreciate my friends, especially my course mates,

prominent among, who we shared knowledge and thought together, thank you so

much.

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 TABLE OF CONTENTS
TITLE PAGE i

DEDICATION ii

DECLARATION iii

CERTIFICATION iv

ACKNOWLEDGEMENT v

TABLE OF CONTENTS vi

LIST OF TABLES ix

CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study 1

1.2 Statement of the problem 4

1.3 Justification 5

1.4 Aim of the Study 6

1.4.1 Objectives of the Study 6

CHAPTER TW0

2.0 LITERATURE REVIEW 7

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2.1 The use of lactic acid bacteria as starter cultures 10

2.1.1 Lactic Acid Bacteria Fermentation 12

2.1.2 Acetic Acid Bacteria fermentation 17

2.1.3 Different Fermented Products 17

2.2 Probiotics 19

2.2.1 Microorganisms used as probiotics 19

2.3 National Shigella Surveillance 20

2.3.1 Infectious Gastroenteritis 22

2.3.2 Causes 24

2.3.3 Clinical Diagnosis 25

2.3.4 Treatment 27

2.3.5 Prevention 28

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Area 29

3.2 Sample Collection 29

3.2.1 Culture media used 29

3.2.2 Sterilization method 30

3.3 Preparation of media 30

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3.4 Purification of culture and preservation 30

3.5 Serial Dilution process 31

3.6 Identification of isolates 31

CHAPTER FOUR

4.0 RESULTS

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion 36

5.2 Conclusion 39

5.3 Recommendations 39

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9
 ABSTRACT

Lactic corrosive microscopic organisms (LAB) are well known to be fundamental

for food creation and wellbeing upkeep. Assessment of the lactobacillus species
disconnected from Nunu's cutthroat bond to Shigella spp. is the point of this
examination. On the main campus of the University of Abuja, the Faculty of
Sciences, a study was conducted in the laboratory of the microbiology department.
Nunu, or matured milk items, were accessible at the College of Abuja's principal
grounds. They were then transported in a clean, sterile container to the laboratory
for microbiological examination in an aseptic manner. Agar (20 ml for every plate)
was ready, and the LAB strains' adherence to chicken epithelial cells was
researched. Chicken ileum was separated aseptically, cleaned, and isolated into
four tiny (1 cm) pieces. Every part was treated in a cell suspension of LAB strains
(10° fu/mL in PBS) at 37°C for an hour and a half. The colony forming unit (cfu/g)
was used to determine the amount of viable bacteria cells in a sample, and simple
descriptive statistics were used to identify the isolated bacteria. The freshness of
the Nunu milk samples was checked, and it was found that their color and flavor
met the requirements for consumption. Eleven provinces altogether were arbitrarily
picked for streaking from the different examples after various weakenings. Six
disconnects were then recognized utilizing morphological, physiological, and
biochemical elements. The discoveries showed that each seclude of Salmonella that
was inspected under a magnifying instrument showed up as short chains of Gram-
positive cocci. The study successfully distinguished and identified Lactobacillus
species from Nunu, shedding light on the microbial makeup of this traditional
fermented beverage. revealing information about these microbes' potential
antibacterial properties. The findings improve our understanding of how
Lactobacillus species might prevent Shigella species from spreading, which is
important information for the food industry and public health.

10
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

It is all around perceived that lactic corrosive microbes (LAB) are significant for

keeping up with wellbeing and creating food (Emiliano et al., 2014). Interest in

lactic corrosive microbes (LAB) with probiotic characteristics and their

antimicrobial activity against pathogenic microorganisms was ignited by the chase

after food fixings with important bio-dynamic characteristics (Hugo, 2006).

Antimicrobial compounds like diacetyl, organic acids, hydrogen peroxide, and

bacteriocins can be produced by lactic acid bacteria (LAB), according to Sezer and

Güven (2009). These compounds can prevent the growth of pathogenic

microorganisms. Privately matured milk is a food handling technique that utilizes

organisms, like lactic corrosive microbes (LAB), to create food through the

maturation interaction (Olanrewaju et al., 2009). In food handling, aging is the

anaerobic utilization of yeast and additionally microbes to change starches over

completely to liquor, carbon dioxide, or natural acids (Williams and Dennis, 2011).

One of the conventional ways of safeguarding food is by maturation. Food of

variable quality is produced when simple, non-sterile equipment, chance or natural

inoculums, uncontrolled conditions, sensory fluctuations, poor durability, and

11
unsightly packaging of processed products are used. Aging procedures are in many

cases limited scope and family based (Olanrewaju et al., 2009).

The method of making privately aged milk includes assembling new cow milk and

allowing it to mature for a couple of days. The Fulanis use calabashes, or elastic

pails, to mature the milk. Locally fermented milk, also known as unu, has a strong,

acidic flavor similar to that of yoghurt. It can be consumed by itself or mixed with

fura and sugar. Indeed, even after the milk is aged, these creatures could in any

case be available in the completed item because of the maturation cycle and the

subsequent pH decrease. It has been laid out that salmonellae and different

organisms feature the meaning of endlessly milk items as a wellspring of human

contaminations.

As per Jara et al. ( 2011), lactobacillus is viewed as a part of the normal microbiota

of the mammalian gastrointestinal framework as well as various different

specialties and matured food varieties. Calcium, riboflavin, phosphorus, vitamins A

and D, and pantothenic acid are all found in high quantities in locally fermented

milk. It additionally has a superb blend of protein, fat, and starches. There are

claims that aging upgrades the milk proteins' assimilation. A hereditarily and

physiologically differed gathering of bar molded, Gram-positive, nonspore-

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framing, nonpigmented, catalase-negative, microaerophilic, to stringently

anaerobic microbes make up the Lactobacillus sort.

The primary properties that are thought to be effective are the modulation of the

immune system, the production of antimicrobial substrates, and the reduction in

serum cholesterol levels. Lactobacillus, otherwise called Lactic Corrosive

Microbes, or LAB, is a financially huge bacterium that has a great many

applications in the food business and as a probiotic specialist to upgrade human

wellbeing (Tannock, 2004). As indicated by Catanzaro and Green (2017), eating an

adequate number of live cells of explicit species and kinds of lactobacilli is

remembered to have various positive physiological impacts on the host, including

bringing down the gamble of digestive problems and protecting a fair and solid

stomach microbiota.

The development of lactic corrosive, which hinders the development of different

microorganisms and permits lactobacilli to set up a good foundation for themselves

in the digestive system, is one of the standards for the in vitro choice of lactobacilli

to be utilized as probiotic fixings in food and drug arrangements that advance

wellbeing (Ammor et al., 2007). Regardless of where they came from, most of

Lactobacillus strains have somewhere around one local plasmid and much of the

time more. A few lactic corrosive microbes may likewise convey anti-microbial

13
obstruction qualities encoded in possibly contagious plasmids, and any strains that

do so are considered ill suited to be utilized as probiotics for people or creatures. In

any case, reports have likewise featured the meaning of characteristically safe

strains to anti-infection agents, which might help patients whose typical stomach

microbiota has become imbalanced or fundamentally diminished in amount

because of taking different antimicrobial prescriptions. et al., Liasi (2019). Various

antimicrobial mixtures created by lactobacillus, including bacteriocin, have been

connected to these wellbeing benefits.

"nunu," a locally fermented milk, is only produced in manageable daily quantities

because of its poor storage quality. This locally fermented milk begins to

deteriorate immediately as a result of the lactic and non-lactic microorganisms that

develop during storage and cause contamination. Poor hygienic practices by those

handling the items may put the customer at risk of being exposed to pathogenic

germs because the products do not undergo additional processing before being

consumed. To help general wellbeing, evaluating privately matured milk items for

lactobacillus arrangement by species, cell thickness, and probiotic exercises keeps

on being the acknowledged technique for deciding item quality and attractiveness.

Understanding the microbiological and biochemical modifications achieved by its

handling would without a doubt further develop creation and fitting use on a

greater scale (Ammor et al., 2007).

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1.2 Issue Proclamation

A solid cow's new milk ordinarily has a low microactivity, particularly when the

bacterial burden is under 103 cfu/ml. As indicated by Jara et al. ( 2011), but if the

load is kept at room temperature for a while, it could increase by up to 100 times.

Some of the factors that increase the bacterial activity in raw milk and its products

include the animal's health, the state of the housing area, the type of feed, the water

used on the farm, the milk vessels and storage utensils, and, most importantly, the

hygiene of the milker and handler. It has been seen that the presence of this

pathogenic bacterium in crude milk and its side-effects represents a serious gamble

to human wellbeing, especially for the people who keep on polishing off crude

milk. It additionally brings down the milk's time span of usability. Emergency

clinic obtained microbial contaminations are turning into a significant general

wellbeing worry that can bring about fatalities and extreme dismalness (Chen et al.,

2006). At the point when irresistible or adaptable sicknesses coming about because

of multidrug safe microorganisms emerge, advantageous medicine is essential on

the grounds that consistently endorsed anti-infection agents are presently not

powerful, making the therapy of such diseases testing (Adegoke and Ogunbanwo,

2017). The CDC (2015) says that pathogenic microbes are the main thing that

cause diseases to spread to humans and animals.

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1.3 The Review's Reasoning

Numerous scientists have turned their concentration to lactic corrosive

microorganisms (LAB) as they continued looking for an alternate way to deal with

treating destructive organisms. Various investigations have exhibited that LAB

species produce a scope of synthetic substances with opposing action, and

scientists have utilized food decay microorganisms against pathogenic microbes as

well as hostile microorganisms to lessen plant and human microorganisms

(Adegoke and Ogunbanwo, 2017).

LAB produce a variety of antimicrobial chemicals, such as ethanol, formic acid,

acetone, hydrogen peroxide, diacetyl, and bacteriocins, in order to naturally

outcompete other microbes in the same niche. Additionally, these compounds

enable LAB to preserve food (Oliveira et al., 2008). Therefore, the evaluation of

the lactobacillus species that were isolated from Nunu's competitive adhesion to

Shigella spp. is the goal of this study.

1.4 The Purpose of the Study

The objective of this research is to determine whether a nunu-derived lactobacillus

strain can successfully compete with Shigella strains.

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1.5 The Goals of This Study

1. To isolate and portray Nunu's Lactobacillus species

2. To find out whether Lactobacillus species restrain Shigella species

17
 CHAPTER TWO

2.0 LITERATURE REVIEW

Lactobacillus fermentum has been identified as a potential probiotic (Mikelsaar,

2009). It is a common inhabitant of the human gastrointestinal tract. Two

commercial strains that are used as probiotics are PCC and ME-3. L. Fermentum is

a viable probiotic because of its notable tolerance to bile and pH. When tested

against various pH concentration solutions, Lactobacillus fermentum's ability to

grow and persist for a few hours after being incubated in a pH solution proved its

strong pH tolerance. Furthermore, studies have assessed Lactobacillus fermentum

strains at different bile concentrations and shown that they are extremely resistant

of bile salt (3 g/L). The fact that Lactobacillus fermentum can flourish in these

conditions provides more proof that it can operate as a probiotic (Pan et al., 2011;

Srinu et al., 2013). The ability of Lactobacillus fermentum to reduce cholesterol

has led to its classification as a probiotic. The results of experiments with several

strains of Lactobacillus and broths containing cholesterol showed that

Lactobacillus fermentum had the greatest ability to remove cholesterol. Among the

techniques used by L. Fermentum may remove cholesterol in vivo by absorbing

cholesterol and accelerating the metabolism of cholesterol. Another method is the

incorporation of cholesterol from the host body into the membrane or cell walls.

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This would also increase the resistance of the bacterial cell membranes to

environmental stress. The third strategy is to increase the body's consumption of

cholesterol. L. The market for cholesterol-based bile salt would increase if

fermentum were present because it would prevent bile salt from being recycled and

would accelerate its excretion. To make L. possible. In order to limit the impact of

utilizing antibiotics, fermentum must not have any transferable resistance genes in

order to be classified as a possible probiotic (Zeng et al., 2010).

L. Additionally, it has been demonstrated that fermentum reduces the severity of

chest infections, the quantity of diseases endurance athletes suffer from, the total

number of days they miss owing to respiratory illnesses (Cox et al., 2008), and the

amount of medication they take (West et al., 2009). Additionally, studies have

demonstrated that it reduces the symptoms of atopic dermatitis (Weston and

Halbert, 2005) and increases immunity to influenza vaccinations (French and

Penny, 2009). Reid (2008) reports that some strains have been utilized to treat

female urogenital infections, while others have been connected to the metabolism

of cholesterol (Mikelsaar and Zilmer, 2009).

Consuming probiotic-rich fermented foods has many other benefits. Fermentation

converts the difficult-to-digest lactose from milk to the more easily digested lactic

acid; it neutralizes anti-nutrients present in many foods, such as the phytic acid
19
found in all grains; it produces new nutrients like omega-3 fatty acids and increases

the levels of folic acid, pyroxidine, B vitamins, and riboflavin in foods. Probiotic

foods also help to strengthen the immune system by producing more antibodies to

fight infectious diseases; and the flora in them forms a barrier that covers the inner

lining of the small intestine and helps inhibit pathogenic organisms like

Salmonella, E. Coli, and an unhealthy overgrowth of Candida. According to

scientific estimates, for a probiotic to have the desired effect, each gram of yogurt

or beverage must contain at least one million of these bacteria (Shiby and Mishra,

2013).

In America, Europe, and the Orient, there is technology available for the

commercial production of several types of probiotic meals. Nonetheless,

individuals in many parts of Africa make probiotic-containing foods on a small

scale. These include dairy products like Nunu and non-dairy ones like Togwa.

Fufu liquor, akamu souring water, and fermented raffia palm sap (Adebolu et al.,

2007; Prado et al., 2008). Most probiotics have been proven to grow successfully

in a dairy-based matrix because of the lactose-hydrolysing enzyme and proteolytic

system involved in casein utilisation, which provide probiotic cells with a carbon

source and essential amino acids for proliferation. The metabolism of these

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nutrients forms organic compounds required for the production of flavor,

preservation, and appearance of the products (Yeo, 2011).

2.1 Establishing lactic acid bacterial colonies

Because of unsanitary cooking conditions, bacteria have been found in many

fermented local dishes from Africa. These come from the raw components or from

the handlers. For these pathogens to proliferate in fermented foods and potentially

cause foodborne illnesses, they need to overcome barriers such as low pH, low

water activity (aw, in solid-state fermentation), and occasionally heat treatments

and naturally occurring antimicrobial chemicals (Gadaga et al., 2004). Foodborne

bacteria can multiply to high quantities during the early stages of fermentation due

to the low acid levels. A few methods to lower the risk of consuming fermented

foods-related foodborne illnesses include the use of protective cultures, starter

cultures, and improved hygiene. Starter cultures are microbial culture preparations

that aid in the initiation of fermentation while preparing various foods and

fermented beverages, according to Farnworth and Mainville (2003).

Lactic acid bacteria starter cultures are employed in the fermentation process to

ensure rapid bacterial growth and a pH drop to below 4, which is crucial for

treating infections (Gadaga et al., 2004). Lactic acid bacteria (LAB) are a clade of

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Gram-positive, low-GC, acid-tolerant, usually non-sporulating, non-respiring rods

or cocci that are related by similar physiological and metabolic characteristics.

These bacteria are usually found in lactic products and decomposing plants. They

produce lactic acid, which is the principal metabolic consequence of the

fermentation of carbohydrates. LAB and food fermentations have historically been

linked to this trait.

The production of organic acids, bacteriocins, a reduction in antinutritive factors,

and the breakdown or inactivation of toxins are among the advantages of lactic

fermentation, and they may all improve the safety of foods fermented using this

process. As a result, the LAB are significant among the microbes connected to

traditional fermented foods. Because of their connection to the human

environment, their beneficial interactions with the human digestive system, and the

long history of lactic fermented foods in many cultures, these foods may be

"Generally Regarded as Safe" (GRAS).

A variety of fermented items, such as meat, vegetables, dairy products, and

beverages, are frequently made using lactic acid bacteria (LAB) as starting

cultures. Regretfully, it is now difficult to get commercial starters for small-scale

fermentation of traditional African cuisine. The LAB are advantageous for

maintaining and improving human health in addition to having a large economic

22
impact. Science has demonstrated that probiotics, or lactic acid bacteria, provide

health advantages (Rashid et al., 2007).

2.1.1 Lactic acid bacteria fermentation

The most control over lactic acid fermentation of all the fermentation processes is

the salt concentration. Lactic acid bacteria fermentation is used with natural

microflora or lactic acid bacterial (LAB) cultures to preserve various items, such as

milk, cereals, meat, fruits, and vegetables. This method, which combines acid and

salt with chemical preservation, is applied all around the world (Thokchom and

Joshi, 2012). Lactic acid (LA) fermentation is a common method used to preserve

and improve the nutritional content and sensory qualities of fruits and vegetables.

By maintaining all of the natural plant components, this procedure improves the

food's quality, flavor, and aroma (Bamforth, 2012).

Daha et al. (2005) claim that LA fermentation enhances the organoleptic and

nutritional properties of the fermented fruits and vegetables while preserving the

nutrients and colorful pigments. At least five goals are served by fermentation in

the food processing industry:

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I. the expansion of food's flavor, aroma, and texture spectrum, which enhances the

diet of humans.

ii. the large-scale food preservation processes including lactic acid, acetic acid,

alcohol, and high salinity; iii. biological enrichment of food substrates with

essential amino acids, essential fatty acids, vitamins, and protein;

IV. Detoxification during food fermentation.

v. Shorter cooking times and less fuel use (Steinkraus, 2006)

Lactic acid fermentation is used throughout the world to produce foods that are not

possible to manufacture with traditional methods. The most commercially relevant

genus of lactic acid fermenting bacterium is Lactobacillus, while other bacteria and

even yeast are used infrequently. Fermenting lactic acid is most commonly used to

make yogurt and sauerkraut. Lactic acid is produced in the goods as a result of

spontaneous fermentation of the sugar in the fruit sample. Lactic acid bacteria are a

family of gram-positive, non-spore-forming cocci or rods that are mostly

recognized for their ability to produce lactic acid as a byproduct. Foods are mostly

preserved by the production of acid, mainly by LAB, which lowers pH to a level

where most rotting microorganisms cannot flourish. (Pederson, 2011).

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Food substrates that are teeming with good, edible microorganisms become

resistant to the invasion of germs that can ruin or poison food. It is hard for less

desirable or destructive species to compete. Consuming fermented fruits and

vegetables from LA contributes to better human nutrition in many ways, such as

promoting a balanced diet, providing vitamins, minerals, and carbohydrates, and

preventing various illnesses like diarrhea and cirrhosis of the liver, all thanks to

their probiotic properties. The relatively new term "probiotic" (meaning "for life")

is usually used to describe microorganisms that are beneficial to humans (Yamano

et al., 2016).

Probiotics are live microorganisms such as Lactobacillus plantarum, L. Casei, L.

food supplements that provide acidophilus and Streptococcus lactis to the host in

order to promote intestinal balance (Tamang et al., 2006). Numerous studies have

connected probiotic supplements to a range of health advantages, such as reduced

blood cholesterol, stronger immune systems, improved gastrointestinal function,

and a lower risk of colon cancer (Anandharaj et al., 2014). Numerous studies have

demonstrated the potential benefits of probiotics in the treatment of a wide range of

illnesses, such as colitis, lactose intolerance, diarrhea, blood pressure, cholesterol,

immune system function, infections, and poor mineral absorption. Significant

probiotic microbes include

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Pediococcus acidilactici, Saccharomyces boulardii, Lactobacillus plantarum,

Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, and

Lactobacillus rhamnosus (Holzapfel et al., 2018).

The genus Lactobacillus is a broad group of LAB with important applications in

the fermentation of food and feed. Probiotic inoculants and meal starters for

fermented foods are both made with lactobacilli. Lactic acid is fermented by

grampositive organisms belonging to the genus Lactobacillus, which is a subgroup

of the wider LAB group. Other genera such as Lactococcus, Enterococcus,

Oenococcus, Pediococcus, Streptococcus, Leuconostoc, and Lactobacillus are also

included in the LAB group since they are capable of producing lactic acid.

The LAB have an advantage over other species that are less resistant to salt since

they can tolerate large quantities of salt. (Anonymous, 2007) They can also

produce acid, which inhibits the growth of undesirable microorganisms.

Leuconostoc sp. is also widely known for starting most lactic acid fermentations

because of its strong tolerance to salt. The salt in the pickles encourages

Leuconostoc sp. development. and LAB while preventing gram-negative bacteria

from growing.

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 LAB is one of the important bacteria in food fermentation, and serological

techniques and 16'S ribosomal RNA cataloging have shown that they are related

phylogenetically and have several traits in common. Lactic acid fermentation

extends the shelf life of fruits and vegetables and enhances their flavor and

nutritional value while reducing toxicity. LAB are well-known for their

fermentative ability, which enhances food safety, organoleptic properties, nutrient

enrichment, and general health advantages (Anonymous, 2007).

Fermented foods have the potential to be a beneficial source of probiotics since

they include lactic acid bacteria, such as Leuconostoc mesenteroides.

Bacteria bifidus, L. sp. Pediocccus cerevisiae, Lactobacillus thermophilus,

Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus citrovorum, and

Streptococcus lactis are among the lactobacillus species. A. Staphylococcus aureus,

Saccharomyces cerevisiae, and pallax. There could be niger within a rotten pickle.

Pickle deterioration can also be caused by microbial contamination or oxidation-

related rancidity of the oil. Products made by using lactic acid bacteria's

fermentation method include:

a. Lactic acid bacteria (LAB) ferment the sugar present in fruit and vegetable

components to produce pickles. Because of the high lactic acid concentration of the

27
generated product, only beneficial bacteria that can tolerate the pH of lactic acid

can survive.

b. Sauerkraut is basically fermented cabbage that has been finely diced and

fermented with lactic acid bacteria. Generally speaking, sauerkraut tastes tart and

has a long shelf life.

C. Yogurt: Yogurt is essentially a fermented product made from milk. Yogurt is

primarily made by fermenting milk with lactic acid and safe bacteria. Typically, the

primary bacteria used are Streptococcus thermophiles and Lactobacillus

bulgaricus.

d. Kimchi is basically a Korean dish. This traditional Korean side dish consists of

salted and fermented vegetables, such as napa cabbage and Korean radishes, that

are seasoned with chili powder, onions, garlic, ginger, and jeotgal. Korean food

includes it as a mainstay (Holzapfel et al., 2018).

2.1.2 Bacteria that Produce Acetic Acid Ferment

AAB is a type of gram-negative bacteria that produces acetic acid by fermenting

ethanol or carbohydrates. The family Acetobacteraceae, which comprises acetic

28
acid bacteria, is composed of ten genera. Businesses use a range of acetic acid

bacteria species to make certain foods and chemicals. Vinegar is produced when

acetic acid bacteria are added to alcoholic liquids. This process produces vinegar as

a byproduct of oxidative fermentation. This procedure involves some aerobic

exertion. Weakly fermented liquors often become sour when they come into

contact with air. This is the outcome of the alcohol being transformed into acetic

acid. In the presence of Acetobacter bacteria, a range of substrates, including

starchy solutions, sugar solutions, and alcoholic meals like wine or cider, ferment

to produce acetic acid, according to Holzapfel et al. (2018). Acetobacter aceti,

which yields 14% acetic acid, is commonly used to make vinegar. CH3COOH is

the product of alcohol + oxygen + starchy solution + sugar solution.

2.1.3 A Range of Fermented Products

Fermented food has been highly valued by people from the dawn of time.

Fermented foods have amazing flavor profiles and are also a good source of

probiotics. The fermentation process itself produces the waste products that are

formed when pyruvate is reduced to regenerate NAD+ in the absence of oxygen.

Bacteria usually create acids through fermentation. Vinegar is directly produced by

bacterial metabolism and contains 5% acetic acid. Bacteria produce acetic acid

during the vinegar-making process. When the casein in milk coagulates due to the
29
acid, curds are produced. Because of the salt, only the lactic acid bacteria are able

to survive in pickling.

Actually, the pickling material's sugar is converted by the LAB to lactic acid. The

acid that is consequently produced shields the food against pathogenic and

putrefactive microbes. Glucose (C6H12O6) is precisely broken down into two

molecules of carbon dioxide (CO2) and two molecules of ethanol (C2H6O5)

during the fermentation process. Pyruvate is transformed into carbon dioxide and

ethanol through the process of ethanol fermentation. This is a crucial phase in the

making of wine, beer, and bread. A ferment with a high pectin concentration has

the potential to produce minute amounts of methanol. Usually, just one of the

products is desired; in the case of bread, the alcohol is baked out, and carbon

dioxide is released into the atmosphere during the alcohol-making process.

Lactic acid fermentation occurs when pyruvate is broken down into lactic acid in

the presence of lactic acid bacteria. This happens in the muscles of an animal when

the muscles need energy more quickly than the blood can supply it. It frequently

occurs after a strenuous workout. Both bacteria and fungi experience it. According

to Holzapfel et al. (2018), this specific type of bacteria transforms lactose into

lactic acid, which gives yogurt its sour taste.

30
2.2 Probiotics

The name "probiotics" was first used by Lilly and Stillwell (1965) to describe

compounds produced by bacteria that promote growth. The word "probiotic" is

derived from a Greek phrase that means "pro-life." Probiotics are living bacteria

that are present in food that, when consumed at certain nutritional levels, balance

the microbiota in the gastrointestinal tract and improve health (FAO/WHO, 2001).

According to these definitions, eating probiotics is good to the host's health

(Vandenplas, 2015). The writings of Metchnikoff and Tissier provide the first

scientific suggestions for the use of bacteria as probiotics. Conversely, research

trials on the effects of probiotics on constipation began in the 1930s

(KoopHoolihan, 2001).

2.2.1 Microorganisms make up probiotics.

The two genera Lactobacillus and Bifidobacterium are most frequently used in

probiotic supplements for humans. Among the other species employed are those

belonging to the genera Enterococcus and Lactococcus (Vandenplas et al., 2015).

The Lactobacilli are thought to be naturally occurring microorganisms that live in

the small intestine since they emerge during the first week of life. Lactic acid

31
bacteria (LAB) have been shown to be effective as probiotics for both human and

animal use (Savadogo et al., 2006).

Among other Bifidobacterium species, certain strains of Bifidobacterium animalis

Bifidobacterium lactis, Bifidobacterium bifidum, Bifidobacterium breve, and

Bifidobacterium longum biotypes infantis and longum are commonly found in

probiotic products together with other lactic acid bacteria (Masco et al., 2005).

It is common practice to isolate Bifidobacterium species from human, animal, and

avian excrement. Large amounts of these are also present in breastfed newborns.

Because bifidobacteria share many metabolic characteristics with lactic acid

bacteria (LAB), including being fermentative and producing lactate among other

acids, they are commonly mentioned when talking about probiotics

(Vankerckhoven et al., 2008). Since the effects of probiotics are strain-specific, it is

crucial to keep in mind that strain identification is required in order to assess the

suitability and effectiveness of probiotics for industrial application. For example,

phenotypic testing and molecular methods for genetic identification are combined

to achieve this. DNA/DNA hybridization and 16S RNA sequencing.

2.3 National Shigella Surveillance

32
It is believed that Shigella causes 500,000 illnesses annually in the

In the US, there were 38 deaths and nearly 5,400 hospital admissions (Scallan et

al.,

2011). The majority of Shigella infections cause gastroenteritis, which manifests as

fever, diarrhea, and stomach cramps and happens a day or two after contact with

the germs. The diarrhea is frequently bloody. Information is primarily passed from

one person to another. Shigella infections can also arise from eating contaminated

food or from handling, drinking, or swimming in contaminated water.

Data are gathered for the national Shigella surveillance program by passively

monitoring human Shigella infections confirmed in laboratories. State and

territorial public health laboratories receive Shigella isolates from clinical

diagnostic laboratories for purposes of subtyping, speciation, and confirmation.

Unusual or untypable isolates are sent to the National Shigella Reference

Laboratory, which is housed in the Centers for Disease Control and Prevention's

(CDC) Enteric Diseases Laboratory Branch (EDLB). Public health laboratories

receive reports from the CDC about its findings.

33
State and territorial public health laboratories report Shigella infections to the CDC

electronically using a variety of techniques. Data is compiled into the

Laboratory Enteric Disease Surveillance (LEDS) system. The Division of

Foodborne, Waterborne, and Environmental Diseases (DFWED) of the National

Center for Emerging and Zoonotic Infectious Diseases is responsible for national

Shigella surveillance in LEDS. The annual overview of this data serves as the

primary national source for information on Shigella species and subtypes. Even

though the government is notified of every Shigella infection, many cases are

likely unreported for a variety of reasons.

Scallan et al., 2016.

Not every patient with a Shigella infection seeks medical attention, and doctors

may neglect to perform the necessary diagnostic tests in the clinical diagnostic

laboratory or get a sample for a laboratory diagnosis. There are varying numbers of

isolates by state and year that lack or have ambiguous species and subtype

information.

2.3.1 Infectious Gastroenteritis

34
An inflammation of the stomach and intestines, typically caused by a viral

infection or bacterial toxins, is referred to as gastroenteritis. It is typified by

symptoms like nausea, vomiting, diarrhea, and cramping. There are two primary

mechanisms that lead to acute gastroenteritis. These are:

a) Damage to the villous brush border of the intestine, which results in osmotic

diarrhea and malabsorption of the intestinal contents

b) Toxins that attach themselves to certain enterocyte receptors and cause the

release of chloride ions. The primary cause of bacterial gastroenteritis is as follows:

a) Increased fluid secretion in the small intestine's proximal region brought on by

low-invasive bacteria or enteropathogens that express luminal toxins.

b) Cytotoxicity or inflammation-induced damage to the mucosa of the colon or

ileum, which may manifest as pus or blood

c) The bacteria's penetration of the mucosa into the reticuloendothelial system, as

observed in typhoid illness (Guerrant et al., 2001).

Over 1.7 billion causes of diarrheal sickness have been identified globally,

accounting for an estimated 2.2 million deaths from the illness each year. In

35
developing countries, diarrheal disease is a major concern due to tainted water

sources, malnourishment, and poor sanitation. For children under five, diarrheal

illness is especially serious in those countries. Malnourishment brought on by

recurrent episodes of diarrhea raises the risk that such kids will get infectious

diarrhea. Thus, there is a greater chance of delayed cognitive development and

stunted growth (Fischer et al., 2012).

In the United States alone, there are claimed to be between 211 and 375 million

cases of diarrheal diseases, 18 million hospital admissions, and 3,100 fatalities

annually. Vomiting and diarrhea are typical signs of gastroenteritis, which is

primarily brought on in children by the Rotavirus virus. The norovirus, usually

associated with winter vomiting or bacterial food poisoning, is the cause of adult

cases. The American College of Gastroenteritis (ACG) and the Infectious Diseases

Society of America (NSA) define diarrhea as the passing of three or more loose or

liquid stools in a single day. Diarrhea that lasts longer than 14 days but less than a

month is considered persistent, and diarrhea that lasts more than 30 days is

considered chronic, according to Humphries and Linscott (2005).

Diarrhea has a noninfectious etiology but can be infectious, meaning it can be

caused by bacteria, viruses, or parasites. It is growing increasingly common in

high-income countries. Diarrhea is mostly caused by dietary intolerances,

36
medication reactions, and gastrointestinal disorders such Crohn's disease,

ulcerative colitis, and celiac disease in wealthy countries. As previously shown,

laboratory tests for infectious etiology, such as a bacterial stool culture, can assist

in making a definitive diagnosis of both infectious and non-infectious

gastroenteritis.

When diagnosing gastroenteritis clinically, numerous symptoms are often taken

into consideration. Gastroenteritis frequently manifests as stomach pain, vomiting,

indigestion, diarrhea, flatulence, gagging, and cramping in the stomach.

Generalized symptoms that impact the entire body include fever, chills, lethargy,

disorientation, appetite loss, dehydration, and weariness. Moore et al. state that

weakness, headaches, a racing heartbeat, insufficient urine production,and/or

weight loss are other common symptoms.

2005).

2.3.2 Causes

37
While parasitic infections are less common, bacterial or viral infections are the

most common causes of gastroenteritis. The most frequent viruses that cause

gastroenteritis include rotavirus, norovirus, adenovirus, coronavirus, enterovirus,

and yersinia enterocolitica, according to Moore et al. (2005). Bacteria such as

Salmonella, Shigella, Yersinia enterocolitica, Escherichia coli, and Aeromonas

species are the most frequent causes of bacterial gastroenteritis.

Among the parasites associated with gastroenteritis are Cryptosporidium, which

can cause an illness when swimming in polluted water, and accidental ingestion of

water or surfaces if hands are not fully cleansed after using the restroom. Giardiasis

is a parasite infection that can be acquired by cleaning an infected baby's diaper

without first washing your hands, handling infected animals, or drinking

contaminated water. Certain drugs, including antibiotics, and specific materials,

like lead, can cause gastroenteritis in susceptible people. Even though infectious

gastroenteritis usually resolves on its own, there are situations in which it is self-

limiting. Dehydration is the main method that it could lead to significant

difficulties, though.

Infectious gastroenteritis kills 1.45 million individuals globally each year (Moore

et al., 2005).

38
2.3.3 Clinical Diagnosis

Bacterial gastroenteritis is a bacterially-induced digestive condition characterized

by discomfort, nausea, vomiting, fever, diarrhea, and abdominal cramps. People

who are under severe circumstances suffer from an electrolyte imbalance and

dehydration. A clinical diagnosis that excludes other potential explanations of the

symptoms is often made using the patient's medical history. Sigmoidoscopies and

radiographic exams are performed by doctors to rule out the likelihood of an

inflammatory bowel disease.

(such persistent sickness) and pus-filled pockets known as pelvic abscesses

(Ahmed et al., 2012). Viral gastroenteritis can spread through intimate contact with

an infected person, especially while sharing food, water, or eating utensils, or

through contact with contaminated surfaces before mouth-to-mouth contact. The

majority of us refer to viral gastroenteritis as "stomach flu," and the only more

common ailment is the common cold. The pathophysiology of rotavirus is as

follows:

39
The rotavirus attaches itself to the developed electrolytes at the tip of the small

pole and penetrates.

intestinal villi.

b) Cause structural changes in the intestinal mucosa, including villous shortening

and mononuclear inflammatory infiltrates in the lamina propria.

c) This virus induces maldigestion of the intestinal lumen and carbohydrates in the

absence of lactose.

d) Impaired nutrition absorption combined with simultaneous inhibition of water

reabsorption may cause the malabsorption component of diarrhea.

f) The enterotoxin NSP4, released by rotaviruses, activates a calcium-dependent

chloride secretory pathway (Ahmed et al., 2006).

Depending on the origin, symptoms of viral gastroenteritis can range from mild to

severe and appear one to three days after infection. Though they usually go away in

a day or two, symptoms might occasionally continue up to 10 days. Young children

who experience vomiting and diarrhea may develop an electrolyte imbalance,

which is very serious and can be fatal if addressed. A lot of misunderstanding and

40
misdiagnosis arises from the common association of other symptoms such fever,

headache, chills, and tiredness with viral gastroenteritis (Ahmed et al., 2006).

2.3.4 Health Care

Bacterial gastroenteritis is generally treated at home with lots of rest, lots of water

consumption to stay hydrated, regular short meals high in minerals, avoidance of

dairy products, and lots of fruit and fiber-rich foods. Bacterial gastroenteritis often

goes away on its own without treatment. Vomiting and diarrhea can cause

dehydration, so it's critical to stay hydrated at home by drinking lots of liquids,

especially water. Treatment is still essential for instances with symptoms even if

certain bacterial and parasite diseases require specialized anti-infective therapy.

Gastroenteritis is commonly treated with penicillin, cephalosporin, antifolate sulfa

combinations, nitroimidazole, penem, glycopeptide, and monolactam antibiotics

(Ahmed et al., 2012).

When someone throws up or has diarrhea, they lose vital minerals including salt,

potassium, and calcium. Soups and broths can be used to replace minerals and

body fluids. Mineral and fluid replacement solutions are sold at pharmacies.

Someone should be brought to the hospital for intravenous fluids and electrolytes if

they are excessively dehydrated or incapable of consuming adequate fluids.

41
Consult a physician before taking any over-the-counter (OTC) treatment for

bacterial gastroenteritis, as certain OTC medications may make symptoms worse.

Unless prescribed or advised by a doctor, anti-vomiting or anti-diarrhea

medications should be avoided as they may cause the body to become infected.

2.3.5 Avoidance

Numerous strategies exist for preventing infectious gastroenteritis; the following

are some of the most effective ones:

a) Generally speaking, good personal hygiene avoids gastroenteritis.

b) Strict hand washing with soap both after using the restroom and before handling

food.

c) Consistently clean the restroom and toilet, paying specific attention to the taps,

doorknobs, and toilet seat.

d) Thoroughly cleaning hands with soap after coming into contact with animals,

especially farm animals

e) Steer clear of close contact with individuals who have gastroenteritis.

42
f) Cutting raw meat on a different cutting board.

g) Steer clear of raw seafood and pork.

g) Maintain a spotless kitchen and all of its appliances.

i) Properly store food and toss out any spoiled or expired goods.

j) Sticking to bottled water when traveling, particularly in underdeveloped nations.

43
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Study Area

The review was led at the College of Abuja's primary grounds, which is arranged

along Nnamdi Azikiwe Air terminal Street, F.C.T., Abuja, at the division of

microbial science research facility of the workforce of sciences. 3.2 Collection of

Samples Items made from fermented milk Nunu were purchased at the University

of Abuja's main campus. Its land area is 11,824 hectares. The matured milk items

were aseptically conveyed in a sterile and clean compartment to the research

facility for microbiological assessment.

3.2.1 Media culture

44
utilizedFor the development and confinement of Lactobacillus species, Deman,

Rogosa, Sharpe (MRS) agar and stock were used; Pathogenic bacteria were

cultured on Nutrient agar (NA), and Shigella spp. were tested for antimicrobials on

Muller-Hinton agar (MHA).

3.2.2 Sterilization

Every piece of glass used in this study was properly cleaned with soap and rinsed

with water before being sterilized in an autoclave for 15 minutes at 121 degrees

Celsius. When use, immunizing circles were fire disinfected and the work seat was

cleaned with 70% ethanol.

3.3 Setting up the media

As recently referenced, some Agar (20 ml for each plate) was ready, and LAB

strains were analyzed for their capacity to stick to chicken epithelial cells (Rebin,

2015). Chicken ileum was removed in an aseptic manner, cleaned, and cut into

four tiny pieces of one centimeter each. Each component was incubated for ninety

minutes at 37°C in a LAB strains cell suspension (10° fu/mL in PBS). Using the

Mac faland standard, the pathogen liquid suspension and lactobacillus liquid

suspension were prepared in distinct test tubes. One test tube held LAB, and the

45
other contained salmonella. Basically, 9 milliliters of typical saline were implanted

with salmonella from a developed example plate, and the turbidity of the

combination met the Macfaland rule.

3.4 Preservation and cultural

purification Four (4) plates for tissue culture Plate A contained chicken ileum,

sterile water, and two milliliters of lactobacillus. Two milliliters of lactobacillus

and Shigella species were on Plate B. + Salmonella species + Ileum of a chicken.

Plate C contained two milliliters of microbe + sterile water + Chicken ileum. Two

milliliters of lactobacillus, sterile water, and chicken ileum were on Plate D. NO

chicken ileum. To check for adhesion, samples were removed from each tissue

culture plate after a 90-minute incubation period, serially diluted, and plated on

MRS agar plates.

3.5 Serial Dilution

Method Each test tube contained nine milliliters of normal saline. Each plate had

seven test tubes ready, so for Plate A, the test tubes were A1 through A7. Each

example from a tissue culture plate was immunized with 1 milliliter in the related

test tube, and the leftover test tubes were sequentially weakened. As a delineation,

46
1 ml of plate A was taken, and 9 ml of test tube A1 was contaminated, trailed by

shaking. One milliliter of Test tube A1 was then infused into nine milliliters of

Test tube A2. After that, samples from test tubes B (LAB + Salmonella + ileum)

and C (Salmonella + water + ileum) were extracted with a dropper and placed in

that order on MRS Agar.

3.6 Seclude ID

In light of their morphological, biochemical, and physiological attributes,

disconnected LAB were portrayed. All methods for characterizing isolates were

carried out with twenty-four-year-old cultures, unless otherwise specified (Sneath,

2006).

3.7 Looking at Information

How much practical microscopic organisms cells in an example was assessed

utilizing province framing units (cfu/g), and the pervasiveness pace of the

disengaged microbes was tracked down utilizing fundamental elucidating

measurements.

47
 CHAPTER FOUR

4.0 RESULT

Table 4.1.1 Reveal the colony count of Lactobacillus and Shigella Table

4.1.1; Concentrations:

Pathogen and Colonies Lactic Acid Bacteria and Shigella formed

101Many colonies 1.2 x 101

1025 colonies 1.5x 105

10314 colonies 1.8 x 105

10411 colonies 1.5 x 105

1052 colonies 1.2 x 102

48
 106none None

Table 4.2: Biochemical Test Results of Lactic Acid Bacteria (LAB) Isolated
from

Cow Milk Key: (+): Positive Reaction (−): Negative Reaction

Key; LAB = lactobacillus

Sal= Salmonella spp

Isolates Shigella Organisms

LAB 1 25 mm

LAB 2 1 mm

LAB 3 4 mm

49
 Salmonella 1 23 mm

Salmonella 2 9 mm

Salmonella 3 8 mm

Key; LAB = lactobacillus

Sal= Salmonella spp

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion

The Nunu milk samples were inspected for freshness, and it was determined that

their color and flavor satisfied consumption requirements. Table 4.1 presents the

morphological description of the isolates from Nunu. Eleven colonies in total were

randomly chosen for streaking from the various samples after multiple dilutions.

They were selected in light of the colony' distinctive features. Six isolates were

then identified using morphological, physiological, and biochemical features.

The findings showed that LAB1 colonies were round, opaque, convex, pale

yellow, and brilliant. The cell morphology was observed under a microscope to be

50
solitary, Gram-positive coccobacilli. The LAB 2 colonies had gram-positive

coccobacilli cell shape and were solitary, spherical, white, fuzzy, and rough. On

the other hand, LAB 3 colonies were discovered to have single, gram-positive

cells with a coccus morphology, and to be opaque, mucoid, spherical, white, and

irregular. Salmonella 1 colonies were found to have partial hemolysis and greenish

patches surrounding them. Salmonella 2 colonies were found to be creamy, white,

opaque, with larger clear zones than Salmonella 3 colonies, which were

transparent and did not exhibit any hemolysis at all. All of the Salmonella isolates

that were found to be Gram positive could be seen under a microscope as brief

chains of cocci. These reports provided support for the conclusions of (Messaoud

et al., 2013; Aesen et al., 2000).

Table 4.2 presents the results of the biochemical test conducted on the lactic acid

bacteria (LAB) that were isolated from Nunu. Except for isolates of Salmonella,

all species of lactobacillus and Salmonella were found to be urease positive,

catalase negative, indole negative, unable to ferment lactose, and resistant to

sterility (Aesen et al., 2000; O'Sullivan et al., 2002). These findings are consistent

with those of Alipin and Safitri (2016) and Mora and Arioli (2014). Chicken

illeum was used to cultivate the LAB isolates in 2% and 6.5% sterile water. After

three weeks, it was found that while LAB1 grew more quickly in 6.5% of sterile

51
water and pathogen than in 2% of sterile water, LAB3 grew more quickly in 6.5%

of sterile water. In both sterile water concentrations, Lab2 grew just as well. While

Strep1 and Strep3 did not grow as well at a 6.5% sterile water concentration but

performed well at 2%, Sal 2 flourished even at a 6.5% pathogen and chicken ileum

concentration. As demonstrated by their resistance to sterile water and chicken

ileum, these LAB isolates are suitable candidates for probiotic therapy since they

can tolerate bile salt. These results are consistent with those of Menconi et al.

(2014).

All of the lactic acid bacteria isolates were found to produce gas when cultured in

broth media, confirming their ability to produce carbon dioxide (CO2) as an

endproduct of glucose fermentation and supporting the findings of (Messaoudi et

al., 2013). This is one of the beneficial traits of lactic acid bacteria thought to be

used as bio-preservatives.

The comparison of the two lactic acid bacteria in Nunu revealed that the species of

Lactobacillus has a higher dominance (70%) in all samples compared to the strains

of Salmonella (30%). indicator organisms that are used to validate the LAB

isolates' ability to combat Shigella species bacteria. Following careful

examination, the following characteristics of the Shigella organism colonies were

52
identified: they were little, smooth, tall, and had a metallic green sheen. The study

of Brook et al. (2004) is supported by these reports.

Table 4.3 shows the inhibition zones of LAB isolates against Shigella organisms.

Different inhibition zones were observed by each species of LAB isolate against

the Shigella bacterium. Among the Lactobacillus species, LAB 1 had the biggest

zone of inhibition (25 mm) against Shigella, followed by LAB 3 (4 mm), and Lab

2 had the lowest zone of inhibition (1 mm) against Shigella. In contrast, Sal1

displayed a 23 mm inhibition against Shigella, Strep 2 displayed a 9 mm

inhibition, and Strep 3 displayed the least level of inhibition (8 mm) against

Shigella among the isolates of Salmonella.

5.2 Summary

the procedure for separating, describing, and testing Nunu's Lactobacillus species

for their ability to suppress Shigella species. The study successfully distinguished

and identified Lactobacillus species from Nunu, providing important insights into

the microbial composition of this traditional fermented beverage.

The study also looked at the ability of Lactobacillus species to inhibit Shigella

species, which shed light on these microbes' potential antibacterial properties. The

53
findings add to our understanding of Lactobacillus species' possible role in

stopping the spread of Shigella species, which is crucial knowledge for the food

industry and the health of the general public.

The data acquired from this study provides new insights into the microbial

dynamics in traditional fermented foods and offers strategies for harnessing the

beneficial properties of Lactobacillus spp. to improve food safety and human

health.

5.3 Advice

1. Using lactic acid bacteria to avoid microbial contamination is highly

recommended because they are cheap and haven't been proved to have any

unwanted side effects. It is also improbable that microbes will develop resistance.

2. Hazard Analysis and Critical Control Points (HACCP), a quality control unit

and a systematic preventive approach to food safety, should be supported and

promoted.

54
 REFERENCES

Ahmed, D., Hogue, A., Elahi, M.S, Ertz, H.P and Hossain, D (2012) Bacterial

etiology of diarrheal disease antimicrobial resistance in Dhaka, Bangladesh,

2005-2008. Epidemiology Infection 140: 1678-1684.

Ammor, M.S, Belen, F.A and Mayo, B. (2007). Antibiotic resistance in non

enterococcal lactic acid bacteria and bifidobacteria. Food Microbiology.

(24):559-570.

55
Anandharaj, M. and Sivasankari, B. (2014). and Rani, R.P. (2014). “Effects of

Probiotics, Prebiotics, and Synbiotics on Hypercholesterolemia: A Review,”

Chinese Journal of Biology, volume. Article ID 572754, 7 pages,

doi:10.1155/2014/572754

Anonymous and FAO., (2007). “Fermented fruits and vegetables. A global

perspective: Bacterial fermentations”. FAO, Publications, Rome,

Humphries, R.M, Linscott and A.J (2015) Laboratory diagnosis of bacterial

gastroenteritis. Clinical Microbiology Revenue 28(1): 1-29.

Jara, S, Sanchez, M, Vera, R Cofre, J and Catro, E. (2011). The inhibitory activity

of Lactobacillus species isolated from breast milk on gastrointestinal

pathogenic bacteria of nosocomial origin. Anaerobe. 17; (6): 474-177.

Jara, S, Sanchez, M, Vera, R, Cofre, J. and Catro, E. (2011). The inhibitory activity

of Lactobacillus species isolated from breast milk on gastrointestinal

pathogenic bacteria of nosocomial origin. Anaerobe. 17; (6): 474-177.

Liasi, S.A, Azmi, T.I, Hassan, M.D, Shuhaimi, M, Rosfarizan, M Ariff, and A.B.

(2019). Antimicrobial activity and antibiotic sensitivity of three isolates of

lactic acid bacteria from fermented fish product, Budu. Malaysian Journal of

Microbiology. 5:33-37.

56
Messaoudi, S., Manai, M., Kergourlay, G., Prévost, H., Connil, N., Chobert, J. M.,

and Dousset, X. (2013). Lactobacillus salivarius: bacteriocin and probiotic

activity. Food microbiology, 36(2), 296-304

Moore, J.E, Corcoran, D, Dooley, JS.G, Fanning, S, Lucey B, (2005)

Campylobacter

Veterinary Resources 36: 351-382.

Tamang, J. P. Tamang, B. Schillinger, U. Guigas, C. and Holzapfel, W. H.

(2009). “Functional properties of lactic acid bacteria isolated from ethnic

fermented vegetables of the Himalayas”. International Journal Food

Microbiology. 135 28–33.

Tannock, G.W. (2014). A special fondness for Lactobacillus. Applied Environment.

Microbiology. 70: 3189- 3194

57